Methods and compositions for contributing to the treatment of cancers

ABSTRACT

Methods and compositions for contributing to the treatment of cancers, especially ovarian tumors, are disclosed. The methods and compositions utilize an endothelin B agonist (ET B ) to enhance the delivery and resulting efficacy of chemotherapeutic agent(s) (e.g., cisplatin and/or cyclophosphamide).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/911,773, filed on Apr. 13, 2007, and is acontinuation-in-part of U.S. patent application Ser. No. 11/461,961,filed Aug. 2, 2006, which is a continuation-in-part of U.S. patentapplication Ser. No. 11/360,236 filed Jun. 28, 2006 (which claims thebenefit of U.S. Provisional Patent Application Nos. 60/655,656;60/655,654; and 60/655,643; all filed on Feb. 22, 2005), which is acontinuation-in-part of U.S. patent application Ser. No. 10/691,915filed Oct. 23, 2003 which claims the benefit of U.S. provisional patentapplication No. 60/420,960, filed Oct. 24, 2002. The contents of all ofthese applications are incorporated by reference herein in theirentirety.

FIELD OF INVENTION

The present invention relates to methods and compositions for thetreatment of cancers, including solid tumors, with an endothelin agonistand at least one chemotherapeutic agent such as cyclophosphamide orcisplatin.

BACKGROUND OF INVENTION

Chemotherapy with most anticancer agents is complicated by limitations,such as low therapeutic index and host tissue toxicity, which leads tolife-threatening situations and decreases the quality of life for manypatients. Lack of efficacy and significant potential side effects ofstandard chemotherapy regimens are driving the search for novelapproaches to anticancer therapy. One approach to increase theeffectiveness of existing anticancer drugs for cancer treatment is toincrease the uptake of drugs into the tumor thereby increasing thetherapeutic tumor concentration.

Successful treatment of cancers, including solid tumors, remains anunfulfilled medical goal, despite increased understanding of themolecular biology of tumor cells and the availability of an increasednumber of potential therapeutic agents. For example, ovarian cancer isthe fifth leading cause of cancer deaths in women, the leading cause ofdeath from gynecological malignancy, and the second most commonlydiagnosed gynecologic malignancy.

One problem in the treatment of cancers is that an effective dose of awide variety of potential chemotherapeutic agents is restricted by theseagents' non-selective, highly toxic effect on normal tissues. As aresult, many patients suffer from the side effects of chemotherapywithout reaping the benefits of the treatment. For example, thechemotherapeutic agents cyclophosphamide and cisplatin inhibit cellularproliferation and induces apoptosis of tumor cells. The clinical utilityof cyclophosphamide and cisplatin have been hampered, however, by doselimiting toxicities. Thus, there is a necessity to develop more specificand less toxic cancer therapies.

Targeted delivery of chemotherapeutic agents to tumors could have theadvantage of enhancing the benefit of chemotherapeutic agents whileminimizing their systemic toxic effects. Such targeted delivery couldalso serve to lower the required dose of chemotherapeutic agents thuspotentially reducing the unacceptable adverse effects of these agents.One possible way to achieve targeted delivery of chemotherapeutic agentsis to utilize the distinctive features of tumor vasculature.

Tumors greater than a few millimeters in size require a constantnutrient supply, and, therefore, develop their own vascular bed andblood flow (Folkman, Cancer Res, 46:467 (1986)). Without constantnourishment from these developing blood vessels, the tumors becomehypoxic and subsequently die. Recruitment of new vasculature frompreexisting blood vessels is termed “angiogenesis.”

During angiogenesis, tumor blood vessels develop substantiallydifferently from normal vasculature, and have different properties.Single layered epithelial cells are the first hastily formed tumor bloodvessels. These newly formed tumor blood vessels do not have a smoothmuscle layer or innervation. Tumors also incorporate mature bloodvessels that possess all their autoregulatory functions. Mattsson etal., Tumor Blood Circulation, CRC Press, Boca Raton, pg. 129 (1979);Reinhold, Tumor Blood Circulation, CRC Press, Boca Raton, pg. 115(1979); Warren, Tumor Blood Circulation, CRC Press, Boca Raton, pg. 26(1979).

Vascular tone (the degree to which blood vessels are dilated orconstricted) is governed by a host of endogenous factors including H⁺,K⁺, Ca²⁺, pO₂, pCO₂ and nitric oxide (NO), as well as other regulatorysubstances such as endothelin (ET-1). Secombe et al., Landes, Austin,pg. 40 (1994); Luscher et al., The endothelium: modulator ofcardiovascular function, CRC Press, Boca Raton, pg. 61 (1990). ET-1contributes significantly to regulating vascular tone (Yanagisawa etal., Nature, 332:411 (1988)) and investigators have shown an increase inET1 and ET_(B) receptor expression in solid tumors including breastcarcinomas. Alanen et al., Histopathology, 36:161 (2000); Nelson et al.,Cancer Res, 56:663 (1996); Kar et al., Biochem Biophys Res Commun216:514 (1995); Pagotto et al., J Clin Invest, 96:2017 (1995); Yamashitaet al., Cancer Res, 52:4046 (1992); Yamashita et al., Res Commun ChemPathol Pharmacol, 74:363 (1991). Further, stimulation of ET_(B)receptors causes an increase in blood supply to tumors throughvasodilation of tumor blood vessels. The present disclosure takesadvantage of this fact by using ET_(B) receptor agonists to selectivelyincrease blood flow to tumors to enhance the targeted delivery ofchemotherapeutic agents

SUMMARY OF INVENTION

The present disclosure is related to selectively-increasing the uptakeof anticancer agents such as cyclophosphamide and cisplatin, into tumorssuch as ovarian tumors by administering an ET_(B) agonist with at leastone anti-cancer agent. A treatment regimen in accordance with thepresent invention reduces tumor volume, such as ovarian tumor volume, inanimals.

The methods of the present invention contribute to the treatment ofcancer. The methods include administering to an animal in need oftreatment, at least one endothelin B (ET_(B)) agonist and at least onechemotherapeutic agent. The cancers treatable in accordance with thepresent invention include, but are not limited to, solid tumors likeovarian tumors, colon tumors, Kaposi's sarcoma, breast tumors,melanomas, prostate tumors, meningiomas, liver tumors, breast phyllodetumors, brain tumors, neck tumors, lung tumors, and combinations oftumors. The methods of the present invention include mechanisms wherebythe ET_(B) agonist selectively increases the blood supply to the tumorthereby increasing the delivery of chemotherapeutic agent(s) to thetumor. The ET_(B) agonist and the chemotherapeutic agents may beadministered substantially simultaneously (e.g. as a single composition)or sequentially (e.g. the chemotherapeutic agent is administered priorto the ET_(B) agonist or vice versa).

The present invention is also directed to compositions having at leastone chemotherapeutic agent, at least one ET_(B) agonist, and,optionally, at least one pharmaceutically acceptable excipient. TheET_(B) agonist(s) and chemotherapeutic agent(s) may be part of the samecomposition, are provided as separate compositions, or both.

ET_(B) agonists suitable for use in either the methods or thecompositions of the present invention include, but are not limited to,ET-1, ET-2, ET-3, BQ3020, IRL1620 (N-suc-[Glu⁹, Ala^(11,15)]ET-1(8-21)), sarafotoxin 56c, [Ala^(1, 3, 11, 15)]ET-1, and combinations.IRL-1620 is a preferred ET_(B) agonist.

Chemotherapeutic agents suitable for use in either the methods or thecompositions of the present invention include, but are not limited to,adriamycin, camptothecin, carboplatin, cisplatin, cyclophosphamide,daunorubicin, doxorubicin, alpha interferon, beta interferon, gammainterferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan,5-fluorouracil, and combinations. Cisplatin, cyclophosphamide, and theircombination are preferred chemotherapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the tumor volume of ovarian tumor mice (N=6) separatelygiven saline, IRL-1620 (3 nmol/kg), saline+cisplatin (2.5 mg/kg), orIRL-1620 (3 nmol/kg)+cisplatin (2.5 mg/kg) once every three days for atotal of 4 doses;

FIG. 2 depicts the body weight of ovarian tumor mice (N=6) separatelygiven saline, IRL-1620 (3 nmol/kg), saline+cisplatin (2.5 mg/kg), orIRL-1620 (3 nmol/kg)+cisplatin (2.5 mg/kg) once every three days for atotal of 4 doses;

FIG. 3 depicts the tumor volume of ovarian tumor mice (N=6) separatelygiven saline, IRL-1620 (3 nmol/kg), saline+cyclophosphamide (75 mg/kg),or IRL-1620 (3 nmol/kg)+cyclophosphamide (75 mg/kg) once every threedays for a total of 4 doses; and,

FIG. 4 depicts the body weight of ovarian tumor mice (N=6) separatelygiven saline, IRL-1620 (3 nmol/kg), saline+cyclophosphamide (75 mg/kg),or IRL-1620 (3 nmol/kg)+cyclophosphamide (75 mg/kg) once every threedays for a total of 4 doses.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention contribute to the treatment ofcancer. In one embodiment, the present disclosure relates to a method ofcontributing to the treatment of cancer comprising administering anendothelin B (ET_(B)) agonist and at least one chemotherapeutic agent.

As used herein, the terms “treat”, “treatment” and “contributing to thetreatment of” shall mean preventing, retarding the progression or growthof, shrinking, or eliminating a cancer including a solid tumor. As such,these terms include both medical therapeutic and/or prophylacticadministration, as appropriate.

Most chemotherapeutic agents have cytotoxic properties that are targetedto destroy cancer cells, but in the process inflict considerable damageto the body's normal physiological systems. It would be of greatadvantage, therefore, to selectively deliver chemotherapeutic agents tosolid tumors thus helping to avoid these negative effects of cancertreatment.

The angioarchitecture of tumor blood vessels is different from that ofnormal blood vessels. Carmeliet & Jain, Nature, 407:249 (2000).Therefore, the vascular reactivity of tumors differs from that of normaltissue. For example, the administration of nitric oxide donors,nicotinamide and bradykinin agonists modulate blood flow to tumors.Jordan et al., Int J Radiat Oncol Biol Phys, 48:565 (2000); Fukumura etal., Am J Pathol, 150:713 (1997); Hirst et al., Br J Radiol, 67: 795(1994).

Endothelin is a vasoactive substance that modulates blood flow and ispresent in large concentrations in breast carcinoma tissues compared tonormal breast tissue (specifically, endothelin can be present in anamount of about 12 pg/mg in breast carcinoma tissues as compared toabout 0.12 pg/mg in normal breast tissue). Kojima et al., Surg Oncol,4(6):309 (1995); Kurbel et al., Med Hypotheses, 52(4):329 (1999); Patelet al., Mol Cell Endocrinol, 126(2):143 (1997); Yamashita et al., CancerRes, 52(14):4046 (1992); Yamashita et al., Res Commun Chem PatholPharmacol, 74(3):363 (1991). Endothelins are a family of cyclic peptideswith 21 amino acids, comprising three isoforms in mammals, ET-1, ET-2and ET-3. Inoue et al., Proc Natl Acad Sci USA 86:2863 (1989);Yanagisawa et al., Nature, 332:411 (1988). Endothelins exert theireffects by binding to two distinct cell surface receptors, ET_(A) andET_(B). The ET_(B) receptor binds the three peptide isotypes with equalaffinity. In contrast, the ET_(A) receptor binds ET-1 with higheraffinity than the other isoforms. Both receptors belong to the Gprotein-coupled receptor system and mediate biological responses from avariety of stimuli, including growth factors, vasoactive polypeptides,neurotransmitters and hormones. Masaki, J Cardiovasc Pharmacol, 35:S3(2000); Gulati, Preface. Adv Drug Deliv Rev, 40:129 (2000); Gulati etal., Am J Physiol, 273:H827 (1997); Levin, N Engl J Med, 333:356 (1995).ET_(B) receptors, a focus of the present invention, are present on bothendothelial cells (ECs) and vascular smooth muscle cells (VSMCs) and areincreased in breast cancer tissue (including in invasive as well as inductal and lobular breast carcinoma tissue in humans) when compared tonormal breast tissue. Wulfing et al., Oncol Rep, 11:791 (2004); Wulfinget al., Clin Cancer Res, 9:4125 (2003); Alanen et al., Histopathology,36(2):161 (2000). Endothelin acts on ET_(B) receptors to producevascular dilation and increase blood flow to breast tumor tissue. ET_(B)receptors predominating on ECs, produce vasodilatation via the releaseof factors such as prostacyclin and nitric oxide. de Nucci et al., ProcNatl Acad Sci USA, 85:9797 (1988). Because ET-1 produces an increase inblood flow to tumors by stimulating ET_(B) receptors, an ET_(B) receptoragonist can be used to selectively increase blood supply to tumors, thusincreasing the targeted delivery and resulting efficacy ofchemotherapeutic agents.

ET_(B) receptors have been shown in, for example and without limitation,ovarian cancers, myofibroblasts, Kaposi's sarcoma tumor and intratumoralvessels, breast cancers and melanomas. Bagnato et al., Am J Pathol,158:841 (2001); Alanen et al., Histopathology, 36(2):161 (2000); Bagnatoet al., Cancer Res, 59:720 (1999); Kikuchi et al., Biochem Biophys ResComm, 219:734 (1996). Therefore, administration of an ET_(B) receptoragonist in combination with a chemotherapeutic agent can be used tocontribute to the treatment of solid tumors, including, withoutlimitation, ovarian cancer, colon carcinoma, Kapoli's sarcoma, breastcancer, and melanomas.

ET_(B) agonists useful in accordance with the present invention include,without limitation, ET-1, ET-2, ET-3, BQ3020, IRL1620 (N-suc-[Glu⁹,Ala^(11,15)]ET-1 (8-21)), sarafotoxin 56c, [Ala^(1, 3, 11, 15)]ET-1, andcombinations thereof. [Ala^(1,3,11,15)]ET-1 is a linear analog of ET-1in which the disulfide bridges have been removed by substitution of Alafor Cys residues. Saeki et al., Biochem Biophys Res Commun, 179:286(1991). BQ3020 and IRL1620 are truncated linear synthetic analogs ofET-1 and are the most widely used selective synthetic agonists. IRL-1620is a linear ET-analog whose structure is based on the carboxy terminalend of ET-1 and has 120,000 fold selectivity for the ET_(B) receptors.Okada & Nishikibe, Cardiovasc Drug Rev, 20:53 (2002); Douglas et al., BrJ Pharmacol, 114:1529 (1995). IRL-1620 is a highly selective and potentET_(B) agonist, with evidence being reported of its selectivity for theET_(B1) receptor subtype in preference over the ET_(B2) subtype. Brookset al., J Cardiovasc Pharmacol, 26 Suppl 3:S322 (1995).

Chemotherapeutic agents useful in accordance with the present inventioninclude, for example and without limitation, alkylating agents,antimetabolites, hormones and antagonists thereof, radioisotopes,antibodies, as well as natural products, and combinations thereof. Forexample, an ET_(B) agonist can be administered with antibiotics, such asdoxorubicin and other anthracycline analogs, nitrogen mustards, such as,without limitation, cyclophosphamide, pyrimidine analogs such as,without limitation, 5-fluorouracil, cisplatin, hydroxyurea, and itsnatural and synthetic derivatives, and the like. As another example, inthe case of mixed tumors, such as adenocarcinoma of the breast, wherethe tumors include gonadotropin-dependent and gonadotropin-independentcells, the ET_(B) agonist can be administered in conjunction with,without limitation, leuprolide or goserelin (synthetic peptide analogsof LH-RH). Additional non-limiting examples of chemotherapeutic agentsthat can be used with the present invention include cyclophosphamide,cisplatin, adriamycin, camptothecin, carboplatin, daunorubicin,doxorubicin, interferon (alpha, beta, and/or gamma), interleukin 2,irinotecan, docetaxel, paclitaxel, topotecan, and therapeuticallyeffective analogs and derivatives of the same.

It is theorized, but not relied upon herein, that endothelin agonistsstimulate ET_(B) receptors to dilate tumor blood vessels, therebyincreasing blood flow and the resultant delivery of chemotherapeuticagents to the tumor. The increased blood perfusion of tumors caused byendothelin agonists also increases oxygenation of the tissue. Improvedoxygenation can enhance the therapeutic action of chemotherapeuticagents. Endothelin also can have mitogenic properties. The mitogenicactions of endothelin can help increase the action of chemotherapeuticagents, when administered together. The mitogenic action of anendothelin agonist can increase the action of chemotherapeutic agents byimproving their incorporation into dividing cells, thus increasing theirefficacy.

Chemotherapy is frequently indicated as an adjuvant to surgery in thetreatment of a cancer. The goal of chemotherapy in the adjuvant settingis to reduce the risk of recurrence and enhance disease-free survivalwhen the primary tumor has been controlled. Chemotherapy is utilized asa treatment adjuvant for a cancer, frequently when the disease ismetastatic. An ET_(B) agonist, therefore, is particularly useful beforeor following surgery in the treatment of a solid tumor in combinationwith chemotherapy.

In another embodiment of the present method, the cancer is a solidtumor. In another embodiment, the solid tumor is selected from the groupconsisting of an ovarian tumor, a colon tumor, Kaposi's sarcoma, abreast tumor, a melanoma, a prostate tumor, a meningioma, a liver tumor,a breast phyllode tumor, brain tumor, neck tumor, lung tumor, andcombinations thereof. In another embodiment, the solid tumor is ovariantumor.

In another embodiment, the ET_(B) agonist is selected from the groupconsisting of ET-1, ET-2, ET-3, BQ3020, IRL1620 (N-suc-[Glu⁹,Ala^(11,15)]ET-1 (8-21)), sarafotoxin 56c, [Ala^(1, 3, 11, 15)]ET-1, andcombinations thereof. In another embodiment, the ET_(B) agonist isIRL-1620.

In another embodiment, the chemotherapeutic agent is selected from thegroup consisting of adriamycin, camptothecin, carboplatin, cisplatin,cyclophosphamide, daunorubicin, alpha interferon, beta interferon, gammainterferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan,5-fluorouracil, and combinations thereof. In another embodiment, thechemotherapeutic agent is cisplatin, cyclophosphamide, or a combinationthereof.

In another embodiment, the increase in the blood supply to the tumorincreases the delivery of the chemotherapeutic agent to the solid tumor.In another embodiment, the ET_(B) agonist and the chemotherapeutic agentare administered substantially simultaneously. In another embodiment,the ET_(B) agonist and the chemotherapeutic agent are administered as asingle composition. In another embodiment, the ET_(B) agonist and thechemotherapeutic agent are administered sequentially. Thechemotherapeutic agent may be administered prior to the ET_(B) agonistor the ET_(B) agonist may be administered prior the chemotherapeuticagent.

The present disclosure also relates to a composition comprising achemotherapeutic agent, and ET_(B) agonist, and an optional excipient.The ET_(B) agonist and the chemotherapeutic agent are part of the samecomposition, are provided as separate compositions, or both. The ET_(B)agonist may be selected from the group consisting of ET-1, ET-2, ET-3,BQ3020, IRL1620 (N-suc-[Glu⁹, Ala^(11,15)]ET-1 (8-21)), sarafotoxin 56c,[Ala^(1, 3, 11, 15)]ET-1, and combinations thereof. The ET_(B) agonistmay be IRL-1620. The chemotherapeutic agent maybe selected from thegroup consisting of adriamycin, camptothecin, carboplatin, cisplatin,cyclophosphamide, daunorubicin, doxorubicin, alpha interferon, betainterferon, gamma interferon, interleukin 2, irinotecan, docetaxel,paclitaxel, topotecan, 5-fluorouracil, and combinations thereof.

In another embodiment, the chemotherapeutic agent is cisplatin,cyclophosphamide, or a combination thereof and the ET_(B) agonist isIRL-1620.

EXAMPLE

It surprisingly has been discovered that the administration of IRL-1620and at least one anti-cancer agent selectively-increases the uptake ofanticancer agents, e.g. cyclophosphamide and cisplatin, into ovariantumors. A treatment regimen in accordance with the present inventionreduces ovarian tumor volume in animals.

A preclinical efficacy study was conducted on the human ovarian tumormodel in mice. The tumor cells used were SK-OV3 cells purchased fromATCC. The mice were 4-6 week old Athymic (nu+/nu+) female mice. TheIRL-1620 was intravenously-infused slowly (over 30 seconds). TheIRL-1620 was obtained from American Peptides, CA. The cyclophosphamidewas obtained from Cadila Pharmaceuticals, Ahmedabad (Batch No. GW-4005).The cisplatin also was obtained from Cadila Pharmaceuticals, Ahmedabad(Batch No. KL-5005).

A solid tumor was induced in each animal using 3 million viable SK-OV3cells suspended in 200 μl of PBS per animal. After 10 days, whenestablished tumors of 100 mm³ in diameter were detectable, the micerandomly were divided into six groups of 6 animals per group with eachgroup receiving different treatments. The treatment regimens wereadministered via the tail vein every third day for a total of 4 doses.The groups were treated as follows: (1) tumor bearing mice receivedsaline (N=6); (2) tumor bearing mice received IRL-1620 (3 nmol/kg)(N=6); (3) tumor bearing mice received vehicle (saline)+cyclophosphamide(75 mg/kg) (N=6); (4) tumor bearing mice received IRL-1620 (3nmol/kg)+cyclophosphamide (75 mg/kg) (N=6); (5) tumor bearing micereceived saline+cisplatin (2.5 mg/kg) (N=6); and, (6) tumor bearing micereceived IRL-1620 (3 nmol/kg)+cisplatin (2.5 mg/kg) (N=6).

The effect of these treatments on the tumor size was measured using adigital caliper for a total of 12 days after drug administration. Themice were monitored and weighed on every third day. The administereddoses were half of the maximum tolerated dose of cyclophosphamide andcisplatin for the human ovarian xenograft model in nude mice reported inpublished literature (British Journal of Cancer (2000) 83, 921-927). Theanimals were euthanized with pentobarbital 150-200 mg/kg for each mouse.Progression, stasis, partial regression or complete regression of tumorgrowth were measured by tumor load measurements with calipers and wascalculated using the following formula: (3.14/6)×larger diameter×smallerdiameter.

The results of the study are shown in the figures. FIG. 1 depicts tumorvolume of ovarian tumor bearing mice given IRL-1620 (3 nmol/kg) andcisplatin (2.5 mg/kg). Six ovarian tumor bearing mice (N=6) were givensaline, IRL-1620 (3 nmol/kg), saline+cisplatin (2.5 mg/kg), or IRL-1620(3 nmol/kg)+cisplatin (2.5 mg/kg). The dosing schedule was once everythree days for a total of 4 doses. Body weight and tumor volumes weremeasured on every third day through the 22nd day. IRL-1620/cisplatintreated mice showed more reduction in tumor volume compared tosaline/cisplatin treated mice.

FIG. 2 depicts body weight of ovarian tumor bearing mice given IRL-1620(3 nmol/kg) and cisplatin (2.5 mg/kg). Six ovarian tumor bearing mice(N=6) were given saline, IRL-1620 (3 nmol/kg), saline+cisplatin (2.5mg/kg), or IRL-1620 (3 nmol/kg)+cisplatin (2.5 mg/kg). The dosingschedule was once every three days for a total of 4 doses. Body weightand tumor volumes were measured on every third day through the 22nd day.The body weight in all groups was similar.

FIG. 3 depicts tumor volume of ovarian tumor bearing mice given IRL-1620(3 nmol/kg) and cyclophosphamide (75 mg/kg). Six ovarian tumor bearingmice (N=6) were given saline, IRL-1620 (3 nmol/kg),saline+cyclophosphamide (75 mg/kg), or IRL-1620 (3nmol/kg)+cyclophosphamide (75 mg/kg). The dosing schedule was once everythree days for a total of 4 doses. Body weight and tumor volumes weremeasured on every third day through the 22nd day.IRL-1620/cyclophosphamide treated mice showed more reduction in tumorvolume compared to saline/cyclophosphamide treated mice.

FIG. 4 depicts body weight of ovarian tumor bearing mice given IRL-1620(3 nmol/kg) and cyclophosphamide (75 mg/kg). Six ovarian tumor bearingmice (N=6) were given saline, IRL-1620 (3 nmol/kg),saline+cyclophosphamide (75 mg/kg), IRL-1620 (3nmol/kg)+cyclophosphamide (75 mg/kg). Dosing schedule was once everythree days for a total of 4 doses. Body weight and tumor volumes weremeasured on every third day through the 22nd day. The body weight in allthe groups was similar.

Based on these results, it is apparent that the administration ofIRL-1620 alone with, inter alia, cyclophosphamide or cisplatincontributes to the treatment of ovarian cancer tumors by reducing tumorsize. There was a significant increase in the tumor volume in salineonly treated ovarian tumor bearing mice. In this study, IRL-1620 alonedid not significantly affect tumor volume when compared to controls.Animals treated with saline/cisplatin showed a significant reduction intumor volume. However, IRL-1620/cisplatin treated mice showed a greaterreduction in tumor volume than saline/cisplatin treated mice. Animalstreated with saline/cyclophosphamide showed a significant reduction intumor volume. However, IRL-1620/cyclophosphamide treated mice showed agreater reduction in tumor volume than saline/cyclophosphamide treatedmice.

Pharmaceutical compositions containing the described active ingredientsare suitable for administration to humans or other mammals. Typically,the pharmaceutical compositions are sterile, and contain no toxic,carcinogenic, or mutagenic compounds that would cause an adversereaction when administered. Administration of the pharmaceuticalcomposition can be performed before, during, or after the onset of solidtumor growth.

A method of the present invention can be accomplished using activeingredients as described above, or as a physiologically acceptable salt,derivative, prodrug, or solvate thereof. The active ingredients can beadministered as the neat compound, or as a pharmaceutical compositioncontaining either or both entities.

As used herein, the term “prodrug” shall mean compounds that transformrapidly in vivo to a compound useful in the invention, for example, byhydrolysis. A thorough discussion of prodrugs is provided in Higuchi etal., Prodrugs as Novel Delivery Systems, Vol. 14, of the A.C.S.D.Symposium Series, and in Roche (ed.), Bioreversible Carriers in DrugDesign, American Pharmaceutical Association and Pergamon Press, 1987.

The pharmaceutical compositions include those wherein the activeingredients are administered in an effective amount to achieve theirintended purpose. More specifically, a “therapeutically effectiveamount” means an amount effective to prevent development of, toeliminate, to retard the progression of, or to reduce the size of asolid tumor. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein.

A “therapeutically effective dose” refers to that amount of the activeingredients that results in achieving the desired effect. Toxicity andtherapeutic efficacy of such active ingredients can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index, which is expressed as the ratio between LD₅₀ andED₅₀. A high therapeutic index is preferred. The data obtained can beused in formulating a range of dosage for use in humans. The dosage ofthe active ingredients preferably lies within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed, and the route of administration utilized.

The exact formulation and dosage is determined by an individualphysician in view of the patient's condition. Dosage amount and intervalcan be adjusted individually to provide levels of the active ingredientsthat are sufficient to maintain therapeutic or prophylactic effects.

The amount of pharmaceutical composition administered can be dependenton the subject being treated, on the subject's weight, the severity ofthe affliction, the manner of administration, and the judgment of theprescribing physician.

The active ingredients can be administered alone, or in admixture with apharmaceutical carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice. Pharmaceuticalcompositions for use in accordance with the present invention thus canbe formulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries thatfacilitate processing of the active ingredients into preparations whichcan be used pharmaceutically.

As used herein, excipients may be pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral orintranasal application which do not deleteriously react with the activecompounds and are not deleterious to the recipient thereof. Suitablepharmaceutically acceptable carriers include but are not limited towater, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds.

When a therapeutically effective amount of the active ingredients isadministered, the composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable solutions, having due regard to pH, isotonicity,stability, and the like, is within the skill in the art. A preferredcomposition for intravenous injection typically will contain an isotonicvehicle although this characteristic is not required.

For veterinary use, the active ingredients are administered as asuitably acceptable formulation in accordance with normal veterinarypractice. The veterinarian can readily determine the dosing regimen thatis most appropriate for a particular animal.

Various adaptations and modifications of the embodiments can be made andused without departing from the scope and spirit of the presentinvention which can be practiced other than as specifically describedherein. The above description is intended to be illustrative, and notrestrictive. The scope of the present invention is to be determined onlyby the claims.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding equivalents of thefeatures shown and described, or portions thereof, it being recognizedthat various modifications are possible within the scope of the presentinvention claimed. Moreover, any one or more features of any embodimentof the present invention can be combined with any one or more otherfeatures of any other embodiment of the present invention, withoutdeparting from the scope of the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1. A method of contributing to the treatment of cancer comprisingadministering an endothelin B (ETB) receptor agonist and achemotherapeutic agent; wherein said ETB receptor agonist consists ofIRL 1620, and wherein said cancer is an ovarian tumor.
 2. The methodaccording to claim 1, wherein said chemotherapeutic agent is selectedfrom the group consisting of adriamycin, camptothecin, carboplatin,cisplatin, cyclophosphamide, daunorubicin, alpha interferon, betainterferon, gamma interferon, interleukin 2, irinotecan, docetaxel,paclitaxel, topotecan, 5-fluorouracil, and combinations thereof.
 3. Themethod according to claim 1, wherein said IRL-1620 and saidchemotherapeutic agent are administered substantially simultaneously. 4.The method according to claim 3, wherein said IRL-1620 and saidchemotherapeutic agent are administered as a single composition.
 5. Themethod according to claim 1, wherein said IRL-1620 and saidchemotherapeutic agent are administered sequentially.
 6. The methodaccording to claim 5, wherein said chemotherapeutic agent isadministered prior to said IRL-1620.
 7. The method according to claim 5,wherein said IRL-1620 is administered prior to said chemotherapeuticagent.
 8. A composition comprising at least one chemotherapeutic agent,IRL-1620, and an optional excipient.
 9. The composition according toclaim 8, wherein said chemotherapeutic agent is selected from the groupconsisting of adriamycin, camptothecin, carboplatin, cisplatin,cyclophosphamide, daunorubicin, alpha interferon, beta interferon, gammainterferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan,5-fluorouracil, and combinations thereof.
 10. The composition accordingto claim 8, wherein said chemotherapeutic agent is cisplatin,cyclophosphamide, or a combination thereof.
 11. The compositionaccording to claim 8, wherein said chemotherapeutic agent is paclitaxel.